Improved Human Pluripotent Stem Cell Attachment and Spreading on Xeno-Free Laminin-521-Coated Microcarriers Results in Efficient Growth in Agitated Cultures
Alan Tin-Lun Lam, Jian Li, Allen Kuan-Liang Chen, William R Birch, Shaul Reuveny, Steve Kah-Weng Oh.
Biores Open Access, 2015
The authors used different bioreactors up to 2.5 L in scale, and successfully cultured hMSCs using the minimum agitator speed required for complete microcarrier suspension. In addition, we also reported a scaleable protocol for the detachment from microcarriers in spinner flasks where they use a short period of intense agitation in the presence of enzymes such that the cells are detached without being damaged. Here, the same approach has been effective for 15 mL ambrTMbioreactors, 100 mL spinner flasks and 250 mL Dasgip bioreactors. Two types of microcarrier were used, with (laminin-521) and without surface coatings, four different enzymes and three different growth media (with and without serum), a total of 22 different combinations. In all cases after detachment, the cells were shown to retain their desired quality attributes and were able to proliferate. Qasim Rafiq presented this data at BioLamina symposium 2015. It was found that human recombinant laminin LN-521 and recombinant fibronectin were the optimal ECM proteins for microcarrier coating. A serum-free expansion, harvest and xeno, and DMSO-free cryopreservation process, using LN-521 coated microcarriers and FREEZEstemTM cryopreservation medium was then developed which demonstrated > 5 fold increase in hMSC yield.
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